tmpC1E TMP

Unique Drought Resistance Functions of the
Highly ABA-Induced Clade A Protein Phosphatase 2Cs1[W][OA]

Govinal Badiger Bhaskara, Thao Thi Nguyen, and Paul E. Verslues*
Institute of Plant and Microbial Biology, Academia Sinica, Taipei, 11529 Taiwan (G.B.B., T.T.N., P.E.V.);
Molecular and Biological Agricultural Sciences Program, Taiwan International Graduate Program, Academia
Sinica, Taipei 11529 Taiwan and National Chung-Hsing University, Taichung 402 Taiwan (G.B.B., P.E.V.); and
Graduate Institute of Biotechnology, National Chung-Hsing University, Taichung 402, Taiwan (G.B.B., P.E.V.)
Six Arabidopsis (Arabidopsis thaliana) clade A protein phosphatase 2Cs (PP2Cs) have established abscisic acid (ABA) signaling
roles; however, phenotypic roles of the remaining three HAI PP2Cs, Highly ABA-Induced1 (HAI1), AKT1-Interacting PP2C1/
HAI2, and HAI3, have remained unclear. HAI PP2C mutants had enhanced proline and osmoregulatory solute accumulation at
low water potential, while mutants of other clade A PP2Cs had no or lesser effect on these drought resistance traits. hai1-2 also
had increased expression of abiotic stress-associated genes, including dehydrins and late embryogenesis abundant proteins, but
decreased expression of several defense-related genes. Conversely, the HAI PP2Cs had relatively less impact on several ABA
sensitivity phenotypes. HAI PP2C single mutants were unaffected in ABA sensitivity, while double and triple mutants were
moderately hypersensitive in postgermination ABA response but ABA insensitive in germination. The HAI PP2Cs interacted
most strongly with PYL5 and PYL7 to -10 of the PYL/RCAR ABA receptor family, with PYL7 to -10 interactions being relatively
little affected by ABA in yeast two-hybrid assays. HAI1 had especially limited PYL interaction. Reduced expression of the main
HAI1-interacting PYLs at low water potential when HAI1 expression was strongly induced also suggests limited PYL regulation
and a role of HAI1 activity in negatively regulating specic drought resistance phenotypes. Overall, the HAI PP2Cs had greatest
effect on ABA-independent low water potential phenotypes and lesser effect on classical ABA sensitivity phenotypes. Both this
and their distinct PYL interaction demonstrate a new level of functional differentiation among the clade A PP2Cs and a point of
cross talk between ABA-dependent and ABA-independent drought-associated signaling.
Plants have well-developed sensing and signal

transduction systems to respond to water limitation
during drought when soil water potential (Cw) decreases. Accumulation of the stress hormone abscisic
acid (ABA) is both one of the outputs of upstream
stress sensing and signaling as well as a key regulator
of downstream responses (Finkelstein and Rock, 2002;
Cutler et al., 2010). ABA signaling regulates many, but
not all, responses to drought and other abiotic stresses
(Verslues and Zhu, 2005). There are nine clade A protein
phosphatase 2Cs (PP2Cs) in Arabidopsis (Arabidopsis
thaliana; Schweighofer et al., 2004; Xue et al., 2008), of
which six have established functions as negative
regulators of ABA signaling. These ABA signaling
PP2Cs included ABA Insensitive1 (ABI1), ABI2, ABA
1
This work was supported by the National Science Council of
Taiwan (grant nos. NSC 972311B001005 and NSC 1002311B
001008 to P.E.V.) and by an Academia Sinica Career Development
Award to P.E.V.
* Corresponding author; e-mail paulv@gate.sinica.edu.tw.
The author responsible for distribution of materials integral to the
ndings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Paul E. Verslues (paulv@gate.sinica.edu.tw).
[W]
The online version of this article contains Web-only data.
[OA]
Open Access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.112.202408
Hypersensitive Germination1 (AHG1), AHG3/AtPP2CA,

Hypersensitive to ABA1 (HAB1), and HAB2 (Koornneef
et al., 1989; Leung et al., 1994, 1997; Meyer et al., 1994;
Rodriguez et al., 1998; Sheen, 1998; Gosti et al., 1999;
Merlot et al., 2001; Leonhardt et al., 2004; Saez et al.,
2004; Kuhn et al., 2006; Yoshida et al., 2006; Nishimura
et al., 2007). These PP2Cs have redundant functions in
ABA signaling, as double and triple mutants have extreme ABA hypersensitivity of seed germination, stomatal regulation, and gene expression (Rubio et al.,
2009).
The molecular role of ABA signaling clade A PP2Cs
has been afrmed by several laboratories that have
shown that ABI1, ABI2, HAB1, and AHG3 all interact
with the RCAR/PYR/PYL family of ABA receptors
(hereafter referred to as PYLs for convenience). PYLABA-PP2C interaction blocks PP2C activity and allows
downstream targets, such as the Suc nonfermentingrelated kinases group 2 (SnRK2s) and other PP2C substrates, to remain phosphorylated (Ma et al., 2009; Park
et al., 2009; Umezawa et al., 2009; Vlad et al., 2009).
This, in turn, leads to the phosphorylation of ABF/
AREB/ABI5 bZIP family transcription factors and the
activation of ABA-induced gene expression (Fujii et al.,
2009) or the phosphorylation of Slow Anion Channel1
in guard cells (Geiger et al., 2009, 2010; Lee et al., 2009).
Substantial data support this model of ABA-stimulated
PYL-PP2C interaction in ABA signaling, including reconstitution of the pathway in Arabidopsis protoplasts
Plant Physiology, September 2012, Vol. 160, pp. 379395, www.plantphysiol.org 2012 American Society of Plant Biologists. All Rights Reserved.
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Copyright 2012 American Society of Plant Biologists. All rights reserved.
379
Bhaskara et al.
and Xenopus laevis oocytes (Fujii et al., 2009; Brandt

et al., 2012). However, at least one of the clade A PP2Cs
(AHG1) is not inhibited by the PYLs, despite having
ABA-hypersensitive seed germination (Nishimura et al.,
2007; Antoni et al., 2012). Since there are 14 PYL family
members and nine clade A PP2Cs, the number of
possible interactions is large. Many of the possible
PP2C-PYL interactions remain to be experimentally
tested.
The remaining three clade A PP2Cs, Highly ABAInduced1 (HAI1), AKT1-Interacting Phosphatase1 (AIP1;
also known as HAI2), and HAI3, are of uncertain
physiological function and unclear roles in PYL-PP2C
signaling. We will refer to these collectively as the HAI
PP2Cs. Mutants of the HAI PP2Cs do not exhibit ABA
hypersensitivity (Yoshida et al., 2006); however, their
expression is highly induced by exogenous ABA
(Fujita et al., 2009). Interestingly, a number of PP2Cs,
including HAI1 and AIP1, were found to be misexpressed in the eskimo1 freezing-tolerant mutant, which
has constitutively high Pro and solute content (Xin and
Browse, 1998; Xin et al., 2007). HAI1 was identied as
one of several stress-related genes controlled by the
key developmental regulator SCARECROW (IyerPascuzzi et al., 2011). These observations suggest a role
of the HAI PP2Cs in abiotic stress; yet, no stress-related
phenotype has been associated with the HAI PP2Cs. At
the molecular level, the limited information we have
about the HAI PP2Cs comes largely from testing their
interaction with signaling proteins identied in studies
of other clade A PP2Cs (Fujita et al., 2009). Thus,
whether the HAI PP2Cs may have distinct targets and
distinct functions in stress physiology remains uncertain. An exception to this is the role of AIP1 and HAI3
in regulating Arabidopsis K+-Transporter1 (AKT1) as
part of a calcineurin B-like-SnRK3-PP2C complex (Lee
et al., 2007; Lan et al., 2011); however, the signicance
of this interaction in drought response is unclear.
Conversely, there are many drought responses for
which the role of PYL-PP2C regulation is unclear. Two
such phenotypes of long-standing interest are osmoregulatory solute accumulation and low-Cw-induced
Pro accumulation. Pro is essential for growth and redox buffering under low-Cw and salt stress (Szkely
et al., 2008; Szabados and Savour, 2010; Verslues and
Sharma, 2010; Sharma et al., 2011). Pro accumulation is
reduced in both ABA-decient mutants (aba2-1) and
the ABA-insensitive mutants abi1-1 and abi2-1, demonstrating a role of ABA in promoting Pro accumulation (Verslues and Bray, 2006). However, ABA applied
to unstressed plants elicits only a small fraction of the
Pro accumulation seen during low Cw (Sharma and
Verslues, 2010). This shows that Pro accumulation also
requires low Cw-dependent signaling mechanisms in
addition to ABA signaling.
Pro accumulation is one part of osmoregulatory solute
accumulation whereby cellular solute content is adjusted
to maintain appropriate volume (or turgor when the
volume is restricted by a cell wall) and water content.
A decrease in external Cw caused by soil drying is
380
compensated by an accumulation of solutes inside the

plant cell. This is often referred to as osmotic adjustment (Kramer and Boyer, 1995; Zhang et al., 1999).
Osmotic adjustment has a role in drought resistance
(Blum, 2005) by allowing plant cells to maintain turgor
under mild to moderate stress levels and resist excessive dehydration under more severe reductions in Cw.
Osmotic adjustment includes the accumulation of
cytoplasmic compatible solutes, such as Pro, as well
as K+ and organic acids largely compartmentalized in
the vacuole (Morgan, 1984; Sharp et al., 1990; Voetberg
and Sharp, 1991; Zhang et al., 1999). The molecular
mechanisms controlling osmoregulatory solute accumulation are largely unknown.
We found that hai1, aip1, and hai3 mutants had increased Pro and osmoregulatory solute accumulation
at low Cw, while other clade A pp2c mutants had lesser
or no effect. As osmoregulatory solute accumulation
was not affected by ABA, these data suggest a low-Cw
signaling function of the HAI PP2Cs distinct from the
ABA signaling clade A PP2Cs. The HAI PP2Cs had
moderate ABA hypersensitivity, which could only be
uncovered in double or triple mutants. HAI PP2C
double and triple mutants had ABA-insensitive seed
germination, which contrasted with the phenotypes of
other clade A PP2C mutants. The HAI PP2Cs also
interacted preferentially with certain members of the
monomer-type PYLs, with HAI1 having especially
limited PYL interaction. These combined results indicate that the HAI PP2Cs are functionally differentiated from other clade A PP2Cs both in their roles as
negative regulators of a distinct set of low-Cw responses and in the specicity and greater ABA independence of their PYL interactions.
RESULTS
HAI1, AIP1, and HAI3 Are Negative Regulators of Low
Cw-Induced Pro Accumulation
We collected transferred DNA (T-DNA) mutants of

all the clade A PP2Cs (Supplemental Fig. S1), including
the HAI PP2Cs (shaded gray in Fig. 1A). Low Cw-induced Pro accumulation of these lines was tested by
transferring 7-d-old seedlings from control medium to
polyethylene glycol (PEG)-infused plates of a range of
Cw representing mild to more severe stress. HAI PP2C
mutants had Pro levels twice that of the ecotype Columbia (Col) wild type at mild low-Cw severity (20.5
to 20.7 MPa; Fig. 1B) and approximately 40% higher
Pro at more severe low Cw (21.2 MPa). Of the other
clade A PP2Cs, abi1td, abi2td, and ahg1-3 had elevated
Pro, but not to the extent seen in the HAI PP2C mutants. Pro accumulation of ahg3-3, hab1-1, and hab21 did not differ from that of the wild type (Fig. 1C).
The increased Pro of abi1td and abit2td was consistent
with many observations that these PP2Cs regulate a
broad spectrum of stress responses and with previous
data showing that the abi1-1 and abi2-1 dominant
Plant Physiol. Vol. 160, 2012

Function of HAI Clade A PP2Cs in Drought Stress
Figure 1. HAI PP2Cs are negative regulators of

low-Cw-induced Pro accumulation. A, Diagram
showing the positions of the HAI PP2Cs (shaded)
relative to other clade A PP2Cs. The diagram is
adapted from the phylogenetic analysis of
Schweighofer et al. (2004). T-DNA mutants used
for our analyses are described in Supplemental
Figure S1. B, Pro contents of the wild type (Col)
and HAI PP2C mutants. Seven-day-old seedlings
were transferred to PEG-infused agar plates of
the indicated Cw and Pro contents measured 96 h
after transfer. Data are means 6 SE (n = 912)
combined from three independent experiments.
Significant differences of mutant versus the wild
type (P # 0.05) at a particular Cw are marked by
asterisks. Data from twice-backcrossed lines of
hai3-1 are also presented and represent means of
three homozygous lines selected after backcrossing. FW, Fresh weight. C, Pro contents of
other clade A PP2C mutants. The T-DNA mutants
of ABI1 and ABI2 are referred to as abi1td and
abi2td to clearly distinguish them from the dominant negative alleles of these genes. Data presentation is the same as in B. D, Pro content of the
hai1-2 mutant complemented with 35S-YFP:
HAI1. Data from three independent T3 homozygous lines are shown along with the Col wild type
and the hai1-2 mutant. Data are means 6 SE (n =
69) combined from two independent experiments. E, Subcellular localization of YFP:HAI1.
The image is from the root of a representative
35S-YFP:HAI1 transgenic line. Bar = 20 mm.
negative mutants had reduced Pro accumulation

(Verslues and Bray, 2006). These data established the
rst low-Cw phenotype of hai1, aip1, or hai3.
For both HAI1 and AIP1, two independent T-DNA
lines had the same phenotype. We also transformed
hai1-2 with 35S-YFP:HAI1 and found that the transgene could complement the Pro (Fig. 1D) and solute
accumulation phenotypes (see below). The N-terminal
YFP:HAI1 fusion protein was predominantly localized
in the nucleus (Fig. 1E), in agreement with Antoni et al.
(2012) and Fujita et al. (2009) and the presence of nuclear localization signals in the HAI1 sequence (Antoni
et al., 2012). This contrasted with the Golgi localization
of HAI1 reported by Zhang et al. (2012) using stable
expression of a C-terminal fusion protein. We also
generated transgenic plants using a C-terminal HAI1:
YFP fusion construct but could not observe expression
of the fusion protein. As Zhang et al. (2012) did not
report whether their construct complemented the hai1
mutant, it is possible that they observed a low level of
mistargeted protein. The C-terminal fusion may have
interfered with a C-terminal nuclear localization signal
present in several clade A PP2Cs (Himmelbach et al.,
2002). Of the two aip1 mutants, aip1-1 has a 59 untranslated region T-DNA insertion and still produces a
reduced level of AIP1 mRNA (Supplemental Fig. S1B).
However, its phenotype is identical to that of aip1-2
(Fig. 1B; see below), indicating that aip1-1 lacks AIP1
function, possibly through blocked or reduced translation of the mutant AIP1 mRNA. For hai3-1, the highPro phenotype was still observed after the mutant was
twice backcrossed to the wild type and plants homozygous for the T-DNA insertion were reselected (Fig.
1B). These results veried that it was disruption of the
HAI PP2Cs that caused increased Pro accumulation at
low Cw.
Double and triple HAI PP2C mutants also had elevated Pro accumulation (Supplemental Fig. S2); however, they did not show additional effects above those
seen in the single mutants, and the hai1-2aip1-1hai3-1 triple mutant had less effect. This indicated that additional positive regulatory mechanisms may need to
be activated to allow even higher Pro accumulation at
low Cw and that the double and triple mutants may
have pleiotropic effects that prevent higher Pro accumulation.

381
Bhaskara et al.
None of the HAI PP2C mutants had increased Pro in

response to salt stress (Supplemental Fig. S3). The hai12aip1-1hai3-1 triple mutant had slightly reduced salt
stress-induced Pro; thus, for this phenotype, there was
an additive effect of knocking out multiple HAI PP2Cs.
The lack of effect on salt stress-induced Pro accumulation suggested that the HAI PP2Cs have a more
prominent role at low Cw when there is a much higher
accumulation of Pro as part of osmoregulatory solute
accumulation.
HAI1, AIP1, and HAI3 Are Negative Regulators of

Osmoregulatory Solute Accumulation
Compared with the Col wild type, HAI PP2C mutants all had lower osmotic potential (Cs), indicating
more solute accumulation, across several low-Cw severities (Fig. 2A). hai1 mutants had the largest effect: in
the most severe low-Cw treatment, the Cs of hai1-1 or
hai1-2 was 0.5 to 0.6 MPa lower than that of the wild
type. This corresponds to more than a 200 mM difference in solute content and was larger than could be
explained by the 20 to 30 mM difference in Pro content
between the wild type and hai1 (Fig. 1B). As the difference between cellular Cs and Cw (which in this case
should be in near equilibrium with the medium Cw
indicated by the dashed lines in Fig. 2A) is a measure
of turgor, these data suggest that hai1 may maintain a
higher turgor pressure at low Cw. Complementation of
hai1-2 with YFP:HAI1 returned Cs to the wild-type
level (Fig. 2D). For the other clade A PP2Cs, abi1td
and abi2td had decreased Cs only at the most severe
low-Cw treatment, while ahg1-3, ahg3-3, hab1-1, and
Figure 2. Mutants of the HAI PP2Cs have increased osmoregulatory solute accumulation and
fresh weight at low Cw. A, Cs of HAI PP2C mutants measured 96 h after transfer of 7-d-old
seedlings to PEG-infused plates of a range of Cw.
Data are plotted as the osmotic potential versus
the agar water potential measured at the time of
sample collection. The dashed line in each panel
plots where agar Cw = seedling Cs. Points below
the dashed line are consistent with a positive
turgor pressure, as more reduced Cs indicates
greater accumulation of osmotically active solutes. Data are means 6 SE (n = 35) of measurements combined from two to three independent
experiments. Significant differences between the
mutant and the wild type (P # 0.05) are indicated
by asterisks. B, Cs of ABA signaling clade A
PP2Cs. Data presentation is as described for A. C,
Seedling fresh weight (F.W.) measured 96 h after
transfer of wild-type and mutant seedlings to a
high-Cw control (20.25 MPa), an intermediate
Cw (20.7 MPa), or more severe low Cw (21.2
MPa) treatment. Data are means 6 SE (n = 35)
combined from two to three independent experiments. Significant differences between the mutant and the wild type (P # 0.05) are indicated by
asterisks. D, Seedling Cs of hai1-2 complemented
with 35S-YFP:HAI1 (left panel; data of three independent T3 homozygous lines are shown) as
well as p5cs1-4, which has reduced Pro accumulation, and aba2-1, which is ABA deficient
(right panel). Experimental procedures and data
presentation are as described for A. E, Effect of
ABA treatment (5 mM) on Cs. Seven-day-old
seedlings were transferred to ABA-containing
plates, and Cs was measured 10 and 96 h later.
Data are means 6 SE (n = 35) combined from
two independent experiments.
382

hab2-1 did not differ from wild-type Cs at any severity

of low-Cw stress (Fig. 2B). The fresh weight of hai11 and hai1-2 seedlings was greater than that of the
wild type at 20.7 and 21.2 MPa (Fig. 2C), demonstrating that increased solute deposition in hai1 was
able to drive increased water retention. aip1 and hai31 had slightly increased or unchanged fresh weight
compared with the wild type. These data demonstrated
that the higher solute concentrations in the HAI PP2C
mutants were a result of increased solute uptake and
synthesis rather than an indirect effect of decreased
growth or increased water loss, which could concentrate the same amount of solutes in a smaller volume.
These observations suggested several intriguing aspects of HAI PP2C physiological function. First, the
greatly increased osmoregulatory solute accumulation
and increased water content indicated a general effect
on osmoregulation, potentially involving many solutes, rather than a specic effect on Pro. Consistent
with this, we found that p5cs1-4, which lacks D1-Pyrroline Carboxylate Synthetase1 activity and is decient
in Pro accumulation (Szkely et al., 2008; Sharma et al.,
2011), did not differ from the wild type in Cs (Fig. 2D).
Thus, loss of Pro accumulation in p5cs1-4 may be
compensated by additional accumulation of other
compounds, and the HAI PP2Cs affect this overall
osmoregulatory control mechanism. Also, osmoregulatory solute accumulation may be independent of
ABA sensitivity, as HAB and AHG mutants did not
differ from the wild type in Cs. Consistent with this,
the ABA-decient mutant aba2-1 did not differ from
the wild type in Cs (Fig. 2D) either in these experiments or in a previous study (Verslues and Bray, 2006).
In addition, application of 5 mM ABA did not affect Cs
(Fig. 2E), even though it was sufcient to induce ABAregulated gene expression (see below). These observations support a role of the HAI PP2Cs in osmoregulatory
solute accumulation that is distinct from the ABA signaling roles of other clade A PP2Cs.
We also assayed Cs and fresh weight of double and
triple mutants of hai1-2, aip1-1, and hai3-1 and found
that they had decreased Cs while maintaining fresh
weight (Supplemental Fig. S4). The double and triple
mutants had similar or less effect than single mutants,
indicating that pleiotropic effect of the combined hai
mutations may prevent further increases in solute
accumulation.
HAI1 Is a Negative Regulator of Osmotic Adjustment

during Soil Drying
We planted mutant and wild-type plants together

in the same pot to ensure that they interrooted and
were exposed to the same soil moisture conditions
and subjected 30-d-old plants to a 12-d waterwithholding period. Soil Cw decreased from 20.15
MPa to approximately 21.3 MPa during this period
(Fig. 3A, inset). Cs of hai1-2 was lower than that of the
wild type on days 8 and 10 of water withholding
Figure 3. hai1-2 has increased osmotic adjustment during soil drying,

while neither it nor other HAI PP2Cs substantially affect leaf water loss.
A and B, Cs and RWC of the wild type (Col) and hai1-2 before water
with holding (day 0) and 6 to 12 d after the start of water withholding.
The two genotypes were grown together in the same pots to ensure that
they were exposed to the same Cw, and the inset shows the mean soil
Cw at various times after water withholding. Data are means 6 SE (n =
39) from two to three independent experiments. Significant differences between the mutant and the wild type (P # 0.05) are indicated
by asterisks. Photographs of representative plants during soil drying
can be seen in Supplemental Figure S5. C and D, Water loss from
detached leaves of the wild type (Col), HAI PP2C mutants, and an ABI1
T-DNA mutant (abi1td). Data are means 6 SE (n = 36) from two to
three independent experiments. Significant differences between the
mutant and the wild type (P # 0.05) are indicated by asterisks. F.W.,
Fresh weight.

383
Bhaskara et al.
(Fig. 3A). Leaf relative water content (RWC) declined

during soil drying but did not differ between the
wild type and the mutant (Fig. 3B). Thus, hai1-2 had
greater osmotic adjustment, since a given decrease in
RWC was accompanied by greater solute accumulation. Plants of hai1-2 were of similar size and appearance as the wild type (Supplemental Fig. S5),
again indicating that increased solute accumulation
of hai1-2 was not caused by decreased growth or
altered development.
HAI PP2C Mutants Have Little Effect on Leaf Water Loss
HAI PP2C mutants did not differ from the Col wild
type in leaf water loss over the rst 3 h after leaf detachment; however, hai1 mutants had decreased water
loss over 4 to 8 h (Fig. 3C). This late decrease in leaf
water loss was not seen in abi1td, which was assayed
for comparison (Fig. 3C), and may be related to the
increased osmoregulatory solute accumulation of hai1.
The lack of difference in leaf water loss between the
abi1td single mutant and the wild type was consistent
with previous reports (Saez et al., 2004; Rubio et al.,
2009). Double and triple mutants of the HAI PP2Cs
were unaffected in leaf water loss (Fig. 3D). This contrasted with double and triple mutants of the other
clade A PP2Cs, which have greatly decreased leaf
water loss as part of their severe ABA hypersensitivity
(Rubio et al., 2009).
hai1-2 Has Enhanced Expression of Dehydration-Protective

Genes But Decreased Expression of Defense Genes
We conducted microarray analysis of hai1-2, as it

had the most unique and prominent phenotypes. A 96
h, low-Cw (21.2 MPa) treatment was used to identify
differentially expressed genes associated with longer
term low-Cw response, similar to Pro and osmoregulatory solute accumulation. In the Col wild type, we
found 1,474 genes up-regulated (Supplemental Table
S1) and 1,530 genes down-regulated (Supplemental
Table S2) at least 1.5-fold by this longer term low-Cw
treatment. Comparison of hai1-2 with the wild type
at low Cw found 61 genes up-regulated and 76 genes
down-regulated in hai1-2 relative to the wild type
(Supplemental Tables S5 and S6). A smaller number of
genes were altered in hai1-2 relative to the wild type
in the high-Cw control: 17 genes up-regulated and 26
genes down-regulated (Supplemental Tables S3 and S4).
These gene expression changes were further analyzed
for enrichment of specic Gene Ontology (GO) terms
and coexpression-based clustering to identify groups
of similarly expressed genes affected by hai1-2.
Response to water deprivation was the most
signicantly enriched GO term in the genes upregulated in hai1-2 under control or low-Cw conditions
(Supplemental Table S7). Several other terms related to
seed dormancy and germination, cold stress, and GA
384
and ABA signaling were also signicantly enriched.

Clustering analysis identied a group of highly coexpressed genes (Fig. 4A) that included many, but not
all, of the genes up-regulated in hai1-2. This cluster
included the dehydrin XERO1, several late embryogenesis abundant (LEA) proteins including EM1, and
the seed storage protein CRUCIFERIN3. Other hai1-2 upregulated genes included stress-induced NAC domain
transcription factors (NAC019 and NAC040), dehydrin
XERO2, seed germination regulator SOMNUS, and
GAST homolog protein2 (GASA2) and GASA3. Quantitative reverse transcription (RT)-PCR analysis conrmed the increased expression of several of these
genes in hai1-2 (Fig. 4B). Overall, these gene expression
changes were consistent with HAI1 negative regulation of dehydration resistance.
In contrast, systemic acquired resistance and several
other GO categories associated with defense responses,
defense-related metabolism, or cell wall modication
were signicantly enriched among the genes downregulated in hai1-2 (Supplemental Table S8). Clustering
analysis found several clusters of coexpressed genes
(Supplemental Fig. S6), and we used quantitative RTPCR to verify the expression of genes in one of the main
clusters (Fig. 4, C and D). In addition to the pathogen
response genes differentially regulated in hai1-2, TAT3,
PBS3, GLIP1, and GDSL also have defense roles
(Lopukhina et al., 2001; Oh et al., 2005; Nobuta et al.,
2007; Kwon et al., 2009; Okrent et al., 2009). The
contrasting sets of up- and down-regulated genes suggest a role for HAI1 in balancing abiotic stress versus
defense responses.
Despite the increased Pro accumulation of hai1-2, we
did not see any evidence in our microarray data for
altered expression of genes related to Pro metabolism.
Quantitative RT-PCR detected a small increase in expression of the Pro synthesis gene P5CS1 in hai1-2 after
96 h at low Cw but no other differential expression of
the core Pro metabolism genes (Supplemental Fig. S7).
Thus, HAI1 may affect Pro accumulation by unknown
mechanisms other than transcriptional regulation of
Pro metabolism genes.
HAI PP2C Double and Triple Mutants Have Differential

Effects on Germination and Postgermination
ABA Sensitivity
To further dene unique and overlapping functions

among clade A PP2Cs, we examined the ABA sensitivity of seed germination as well as postgermination
(vegetative) ABA sensitivity. In seed germination, HAI
PP2C single mutants did not differ from the wild type
except for a small but signicant ABA-insensitive phenotype observed in hai1-2 (Fig. 5). These results were
in agreement with previous tests of ABA sensitivity
(Yoshida et al., 2006; Guo et al., 2010; Antoni et al.,
2012). Double and triple HAI PP2C mutants had more
pronounced ABA-insensitive germination (Fig. 5), which
contrasted with the strong ABA hypersensitivity of

Figure 4. Abiotic stress-associated genes up-regulated and defense genes down-regulated in hai1-2. A, Cluster of coexpressed
genes up-regulated in hai1-2. Coexpression-based clustering was performed using a database of 3,800 arrays (see Materials
and Methods). Each edge connecting two genes represents a coexpression relationship having a Pearson correlation coefficient
$ 0.75. See Supplemental Tables S3 and S5 for full lists of genes up-regulated in hai1-2 relative to the Col wild type under either
control or low-Cw conditions. UNK, Gene of unknown function. B, Quantitative RT-PCR verification of genes found to be more
highly expressed in hai1-2 at low Cw by microarray analysis. The genes analyzed along with gene descriptions are given in
Supplemental Table S5. Gene expression analysis was performed for Col wild-type (w.t.) and hai1-2 seedlings under control
conditions or 96 h after transfer to low Cw (21.2 MPa). Data are means 6 SE (n = 3) using samples collected from three independent experiments. All gene expression differences at 21.2 MPa were found to be significant (P # 0.05) by one-sided t test.
Note that XERO2, NAC19, and NAC40 are not part of the coexpression cluster shown in A. C, Cluster of coexpressed genes
down-regulated in hai1-2. Analysis was performed as described for A. The full set of coexpressed clusters of genes downregulated in hai1-2 can be seen in Supplemental Figure S6, and the full list of genes down-regulated in hai1-2 relative to the Col
wild type is shown in Supplemental Tables S4 and S6. D, Quantitative RT-PCR verification of genes found to have reduced
expression in hai1-2 at low Cw by microarray analysis. Procedures and data presentation are as described for B. The genes
analyzed along with gene descriptions are given in Supplemental Table S6.
double and triple mutants of other clade A PP2Cs

(Rubio et al., 2009). We also measured the germination
of ABI, HAB, and AHG mutants and found the expected ABA-hypersensitive phenotypes (Supplemental
Fig. S8).
We examined postgermination ABA sensitivity rst
by measuring the portion of seedlings forming green
cotyledons on 0.5 mM ABA, a concentration that had
minimal effect on germination. HAI PP2C single mutants again showed no difference compared with the
Col wild type (Fig. 6, A and B). Double and triple HAI
PP2C mutants showed greater inhibition of cotyledon
growth than the wild type; however, the response of
the hai1-2aip1-1hai3-1 triple mutant was equivalent to
that of the ahg1-3 single mutant (Fig. 6, A and B).
Similar results were obtained in experiments where
root elongation was quantied after transfer of seedlings from control medium to medium containing a
range of ABA concentrations (Fig. 6, C and D). HAI
PP2C single mutants did not show ABA-hypersensitive
root growth inhibition. In fact, root elongation of aip11 and hai3-1 was greater than in the wild type at low
ABA concentrations (Fig. 6C). ABA hypersensitivity

was seen in double and triple HAI PP2C mutants (Fig.
6, C and D), but the effect was similar to abi1td and
abi2td single mutants (Fig. 6E) rather than hab1-1abi12ahg3 or hab1-1abi1-2abi2-2, which had constitutively
reduced root elongation because of their extreme ABA
hypersensitivity (Rubio et al., 2009). HAI PP2C double
and triple mutants also showed enhanced ABA induction of NCED3 and COR15A expression, which was
again similar to the abi1td single mutant (Fig. 6F). Of the
HAI PP2C single mutants, only hai1-2 showed an increase in COR15A expression, and none of the single
mutants affected NCED3 expression. ABA-induced Pro
was also assayed as an additional measure of ABA
sensitivity. Exogenous ABA applied to unstressed
plants elicits a low level of Pro accumulation (Sharma
and Verslues, 2010). Only the HAI PP2C triple mutant
had a small increase in ABA-responsive Pro accumulation (Supplemental Fig. S9). This contrasted with the
robust increase in low-Cw-responsive Pro accumulation seen in single mutants of the HAI PP2Cs (Fig. 1B).
These assays collectively showed that loss of all three

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Bhaskara et al.
Figure 5. HAI PP2C double and triple mutants

have ABA-insensitive seed germination. Germination was scored based on radicle emergence 4
d after the end of stratification. Data are means 6
SE (n = 34) from independent experiments. Significant differences between the mutant and the
wild type (P # 0.05) are indicated by asterisks.
For comparison, germination assays of the ABA
signaling clade A PP2Cs conducted under the
same conditions can be seen in Supplemental
Figure S8.
HAI PP2Cs did uncover ABA hypersensitivity but also

indicated a lesser role of the HAI PP2Cs in ABA sensitivity compared with other clade A PP2Cs.
To determine whether low-Cw-induced ABA accumulation was affected by the HAI PP2Cs, we measured ABA content at 0, 10, and 96 h after transfer to
21.2 MPa for mutants of all of the clade A PP2Cs
(Supplemental Fig. S10; the 10-h time point represents
the peak ABA accumulation, while 96 h is the steadystate ABA level [Verslues and Bray, 2006]). Decreased
ABA accumulation was observed in hab1-1 and ahg3-3.
However, we saw no difference in ABA content of HAI
PP2C single mutants. Only in hai1-2aip1-1hai3-1 at 96 h
did we see an increase in ABA (Supplemental Fig. S10),
the opposite effect to that seen in triple mutants of other
clade A PP2Cs (Rubio et al., 2009). The ABA content
data further illustrated functional differentiation
among the clade A PP2Cs and also indicated that
differences in ABA content were not responsible for
the gene expression, Pro, or osmotic potential phenotypes of the HAI PP2C mutants.
HAI PP2Cs Have a Distinct Pattern of PYL Interaction
One basis for the unique physiological function of

the HAI PP2Cs could be differences in PYL interaction
and regulation compared with other clade A PP2Cs.
Sequence comparison found that several amino acids
shown by structural and mutational analysis (Miyazono
et al., 2009; Nishimura et al., 2009; Santiago et al.,
2009a; Yin et al., 2009; Dupeux et al., 2011) to be important for PYL interaction of ABI1, ABI2, and HAB1
were not conserved in the HAI PP2Cs (Supplemental
Fig. S11). To test the importance of these differences,
we rst performed a set of qualitative yeast twohybrid assays to compare the PYL interactions of
386
HAI1 with those of HAB1, whose PYL interactions

have been characterized previously (Melcher et al.,
2009; Park et al., 2009; Santiago et al., 2009b). We used
full-length HAI1 but an N-terminal deletion of HAB1
(DNHAB1), as full-length HAB1 autoactivated both
in our experiments and in previous work (Saez et al.,
2008).
HAB1 interacted with all of the PYLs except PYL13
(Fig. 7A). Interaction with the dimeric PYLs (PYR1,
PYL1, and PYL2; Dupeux et al., 2011; Hao et al., 2011)
was ABA dependent except for a low level of PYL1
interaction detected without added ABA. PYL4 to -10
have been shown to be monomers (except PYL7, which
is predicted to be monomeric but has not been experimentally tested; Dupeux et al., 2011; Hao et al., 2011).
HAB1 interaction with these PYLs was not dependent
on the addition of ABA (with the exception of PYL4;
consistent with the results of Park et al. [2009]). In
contrast, HAI1 had detectable interaction only with
PYL5 and PYL8 to -10 (Fig. 7A). Also, b-galactosidase
staining could only be seen after much longer incubation times for HAI1 than for HAB1, suggesting that
the HAI1 interactions were weaker than those of
HAB1. There was a weak HAI1-PYL9 interaction without added ABA that could be reproducibly detected in
quantitative assays (see below) but was not seen in
b-galactosidase staining.
For more detailed analysis of HAI PP2C-PYL interactions, we performed quantitative yeast two-hybrid
assays (Fig. 7B) and found that all three HAI PP2Cs
interacted with PYL5 and PYL8 to -10 but had no detectable interaction with the dimeric PYLs (PYR1,
PYL1, and PYL2). HAI1 differed from AIP1 and HAI3
in that it had the most limited range of PYL interaction,
and its PYL interactions were consistently weaker
(note the difference in scale between the top panel of
Fig. 7B and the two other panels). Perhaps most striking

Figure 6. HAI PP2C double and triple mutants have postgermination ABA hypersensitivity similar to that of single mutants of
other clade A PP2Cs. A, Green cotyledon emergence of the wild type, HAI PP2C mutants, and ahg1-3, which is shown for
comparison. Seeds of each genotype were plated on control medium or medium containing 0.5 mM ABA, and photographs were
taken after 8 d of growth. B, Quantification of green cotyledon emergence after 5 d of growth for all genotypes shown in A. Data
are means 6 SE (n = 3) combined from three independent experiments. Significant differences of the mutant versus the wild type
are marked by asterisks. C, Four-day-old seedlings were transferred to the indicated ABA concentration, and root elongation of
Col wild-type and mutant seedlings was measured over the subsequent 7 d. Data are means 6 SE (n = 1015) combined from
three independent experiments. Significant differences of the mutant versus the wild type at a particular ABA concentration are
marked by asterisks. D, Photographs of representative mutant and wild-type seedlings 7 d after transfer to control (no ABA)
plates (top panel) or plates containing 2 mM ABA (bottom panel). E, Root elongation of abi1td and abi2td assayed for comparison
with HAI PP2C data in C. F, Expression of the ABA-responsive genes NCED3 and COR15A in the Col wild type, HAI PP2C
mutants, and abi1td. Seven-day-old seedlings were transferred to control medium or medium containing 5 mM ABA for 10 h.
Data are means 6 SE (n = 3) combined from two independent experiments. Significant differences of the mutant versus the wild
type are marked by asterisks.
was the lack of HAI1 interaction with PYL7, which

strongly interacted with AIP1 and HAI3 (Fig. 7, B and
C). HAI3 had a somewhat broader interaction range
than HAI1 or AIP1 in that it also had detectable interaction with PYL3, PYL6, PYL11, and PYL12 (Fig.
7B). These experiments used an N-terminal AIP1

truncation (DNAIP1, lacking amino acids 1118;
equivalent to DNHAB1), as full-length AIP1 caused
autoactivation. Full-length HAI3 did not cause autoactivation.

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Bhaskara et al.
Figure 7. Differing PYL interactions of the HAI PP2Cs and the ABA signaling PP2C HAB1. A, Colony-lift b-galactosidase
staining assay comparing the PYL interaction of HAI1 and DNHAB1 (N-terminal deletion construct) without ABA or with 10 mM
ABA added to the yeast culture. Note that the colony lifts for HAB1 were incubated for 2 to 3 h while those of HAI1 were
incubated longer (12 h or overnight) to allow the weaker HAI1 interactions to be seen with similar staining intensity. B,
Quantitative yeast two-hybrid assay of PYL interactions of HAI1 (full length), DN-AIP1, and HAI3 (full length) either without
ABA or with 10 mM ABA added to the yeast culture. Note the difference in scale between the top panel (HAI1) and the other two
panels. Insets show selected data replotted using an expanded y axis scale for clarity. Data are means 6 SD of b-galactosidase
activity from three to four independent yeast colonies. C, Repeated quantitative yeast two-hybrid assays testing PYL5, PYL7,
PYL8, or PYL10 interaction with all four PP2Cs in the same experiment. Data are means 6 SD from three to four independent
yeast colonies. Note the difference in scale between the different panels.
We performed several additional experiments to verify these results, particularly the limited PYL interaction
of HAI1. First, we selected several PYLs having differing
PP2C interactions (PYL5, -7, -8, -9, and -10) and again
assayed their interactions with HAB1, HAI1, AIP1, and
HAI3 to ensure that the patterns found in Figure 7, A
and B, could be conrmed when all PP2Cs were tested
in the same experiment. These experiments again found
that the PYL interactions of HAI1 were consistently
weaker than those of the other PP2Cs (Fig. 7C). Interestingly, PYL5 interacted with HAB1 without added
ABA but had no or very weak interaction with HAI1,
388
AIP1, or HAI3 unless ABA was added. PYL7 had weak

or nondetectable interaction with HAB1 and HAI1 but
interacted strongly with AIP1 and HAI3. Interaction
of PYL7, -8, and -10 was little affected by added ABA.
PYL9 had a slightly different pattern, with HAI3
having strong interaction without ABA but HAB1 and
AIP1 having ABA-stimulated interaction (Fig. 7C).
PYL9 interaction with HAI1 was consistently detected
but was much weaker; thus, it was difcult to determine
the effect of ABA on this interaction. For PYL5, -8, -9,
and -10, a range of ABA concentrations were tested,
and HAI1 interaction was always less than that of

HAB1 (Supplemental Fig. S12A). We also constructed

DNHAI1 and found that it had weak PYL interaction,
essentially identical to full-length HAI1 (Supplemental
Fig. S12B). Western blotting demonstrated that the
weak PYL interactions of DNHAI1 were not caused by
differences in protein expression (Supplemental Fig.
S12C). Overall, these data demonstrated a distinctive
pattern of PYL interaction for the HAI PP2Cs, with
varying effects of ABA on these interactions and an
especially limited PYL interaction of HAI1 either with
or without added ABA.
Differential Effects of Low Cw on HAI PP2C and PYL
Expression Further Suggest Minimal PYL Regulation of
HAI1 at Low Cw
Our microarray analysis indicated that the three HAI

PP2Cs were more highly induced by low Cw than any
of the other clade A PP2Cs (Fig. 8A), and this was
conrmed by quantitative RT-PCR (Fig. 8C; note the
differing scales for each graph). Conversely, expression of many PYLs was down-regulated by low Cw in
the microarray analysis (Fig. 8B). Quantitative RT-PCR
conrmed the strong low-Cw down-regulation of PYL5
and PYL8 and the moderate down-regulation of PYL9
in the Col wild type (Fig. 8C). In contrast, expression of
PYL7 and PYL10 was little affected by low Cw. While
gene expression differences are at best imperfect indicators of protein abundance, the down-regulation of
the HAI1-interacting PYLs further suggests a minimal
PYL regulation of HAI1 at low Cw. The steady
expression of PYL7 and PYL10 indicates that they

may be more important in regulating AIP1 and HAI3
at low Cw.
We also assayed PP2C and PYL expression during
low Cw in the ABA-decient mutant aba2-1. Low Cw
induction of the HAI PP2C expression was greatly
inhibited in aba2-1, indicating that their expression was
at least partially dependent on ABA (Fig. 8C). In contrast, repression of PYL5, PYL8, and PYL9 by low Cw
was largely independent of ABA. PYL10, and to a
lesser extent PYL7, were low Cw induced rather than
repressed in aba2-1. As overexpression of PYL5 (Santiago
et al., 2009b) or PYL9 (Ma et al., 2009) leads to ABA
hypersensitivity, the regulation of PYL expression may
be a point of cross talk between ABA-dependent and
ABA-independent signaling factors.
DISCUSSION
Our analysis found both some similarity as well

as prominent differences between the physiological
function and PYL interaction of the HAI PP2Cs compared with other clade A PP2Cs. Single mutants of the
HAI PP2Cs had increased levels of low-Cw-induced
Pro and osmoregulatory solute accumulation. These
phenotypes were seen to a lesser extent in abi1td and
abi2td but not in mutants of other clade A PP2Cs.
Mutants of hai1 also maintained higher fresh weight at
low Cw, indicating that greater osmotic adjustment in
hai1 led to increased water uptake. hai1-2 also had increased expression of a number of drought-protective
Figure 8. Differing patterns of PP2C
and PYL expression at low Cw. A, Effect
of low Cw on gene expression of clade
A PP2Cs in the Col wild type. Data are
from the microarray analysis described
in Figure 4 and are shown as means 6
SD from three experiments. B, Effect of
low Cw on PYL expression in the Col
wild type from the microarray analysis.
ND, Not detected. C, Quantitative RTPCR was conducted on samples 0, 10,
or 96 h after transfer of 7-d-old seedlings to 21.2 MPa low-Cw treatment.
Data are normalized to the expression
level at time 0 for both the Col wild
type and aba2-1 and are means 6 SE
(n = 34) of samples collected from
three or four independent experiments.

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Bhaskara et al.
genes, including dehydrins and LEAs. Overall, the data

demonstrated that the HAI PP2Cs regulate a distinct set
of drought responses related to dehydration resistance
rather than avoidance of leaf water loss, which is a
prominent phenotype of other clade A pp2c mutants.
ABA application to unstressed plants cannot duplicate the effect of low Cw on Pro or osmoregulatory
solute accumulation. Thus, these traits are controlled
by low-Cw-specic signaling, which is attenuated by
the HAI PP2Cs. It is not without precedent to nd
differing effects of low Cw versus exogenous ABA. For
example, the use of ABA-decient mutants has shown
that during low Cw, ABA is a promoter of root growth
rather than an inhibitor (Sharp et al., 1994; Sharma
et al., 2011). Interestingly, low-Cw-induced HAI PP2C
expression was largely dependent on ABA, suggesting
that the HAI PP2Cs are a point of cross talk between
ABA signaling and signaling directly responsive to
low Cw (Fig. 9).
Double and triple hai mutants did exhibit ABA hypersensitivity in postgermination responses to ABA,
including inhibition of cotyledon emergence and root
growth and induction of ABA-regulated genes. This is
consistent with previous ndings (Fujita et al., 2009;
Antoni et al., 2012) that the HAI PP2Cs may participate
in feedback regulation that reduces ABA sensitivity.
However, these ABA sensitivity differences could only
be seen in double and triple HAI PP2C mutants, and
even then they were more similar to single mutants of
the other clade A PP2Cs rather than the severe ABA
hypersensitivity of hab1-1abi1-2ahg3-2 or hab1-1abi12abi2-2 (Rubio et al., 2009). An even more clear contrast
between HAI PP2Cs versus other clade A PP2Cs is that
double and triple HAI PP2C mutants were ABA insensitive in seed germination. Also, none of the HAI
PP2C single, double, or triple mutants had the reduced
ABA accumulation that we observed for hab1-1 and
ahg3-3 and that was even more apparent in abi1ahg3hab1 (Rubio et al., 2009). Thus, the combined data
indicate that the HAI PP2Cs have a more prominent
role in controlling Pro and osmoregulatory solute accumulation, which were clearly affected in HAI PP2C
single mutants, rather than ABA sensitivity, which only
differed in double or triple mutants.
The PYL interaction pattern of the HAI PP2Cs differed dramatically from that of the ABA signaling
PP2C HAB1. The HAI PP2Cs had marked preference
for interaction with monomer-type PYLs. We did not
detect any interaction of the HAI PP2Cs with the dimeric ABA receptors PYR1, PYL1, and PYL2, although
we do not exclude the possibility that these PYLs have
limited ability to inhibit HAI PP2C activity, as suggested by in vitro assays (Antoni et al., 2012). The HAI
PP2C interaction with PYL7 to -10 was unaffected or
only moderately stimulated by added ABA. This is
consistent with previous analysis of monomeric PYLs
(Dupeux et al., 2011; Hao et al., 2011). The relatively
ABA-independent PYL interaction of the HAI
PP2Cs would seem consistent with their prominent
roles in low-Cw-specic signaling regulating Pro and
390
Figure 9. Model of HAI PP2C function during low-Cw stress. Perception of water loss by the plant elicits ABA accumulation that causes the
activity of clade A PP2Cs, such as HAB1, to be inhibited by ABAstimulated PYL interaction. This, in turn, allows SnRK2 kinases to remain
active and phosphorylate targets such as ABF transcription factors
controlling ABA-dependent gene expression and guard cell slow anion
channel (SLAC) ion channels influencing stomatal aperture. Expression of the HAI PP2Cs is induced in a partially ABA-dependent manner. Because of their highly induced expression and limited PYL
interaction, the HAI PP2Cs can remain active and act in feedback
regulation of ABA signaling, possibly through dephosphorylation of
SnRK2s (Fujita et al., 2009; Antoni et al., 2012). However, the more
prominent role of the HAI PP2Cs, particularly HAI1, is in attenuating
the low-Cw signaling controlling Pro and osmoregulatory solute accumulation. The specific PYL interaction pattern of HAI PP2Cs also
suggests differences in their substrate recognition. Such differential
recognition of substrates in low Cw and ABA signaling may be a basis
for cross talk between these two types of signaling mediated by the
clade A PP2Cs.
osmoregulatory solute accumulation. The specic PYL

interaction of the HAI PP2Cs and differences in their
protein sequences compared with other clade A PP2Cs
also suggest different specicity for dephosphorylation
substrates (for further explanation, see below). HAI1
(as well as AIP1 and HAI3) may have relatively strong
recognition of substrates in low-Cw signaling but
weaker recognition of substrates in ABA signaling (Fig.
9). The converse may be true for other clade A PP2Cs,
which have strong effects on ABA sensitivity. Differing
PP2C substrate specicities could be another basis for
cross talk between low Cw and ABA signaling (Fig. 9).
Implications of the PYL Interaction Pattern of HAI PP2Cs
Recent studies have continued to add complexity

and specicity to the core model of PP2C-ABA-PYL interaction. Examples include both the ABA-independent
PP2C interactions of the monomeric PYLs (Dupeux

et al., 2011; Hao et al., 2011) as well as differences in

PYL regulation of different clade A PP2Cs. For example,
AHG1 lacks the critical Trp for interaction with PYLbound ABA and thus is not PYL regulated (Dupeux
et al., 2011; Antoni et al., 2012). Other clade A PP2Cs
also differ in their PYL regulation both in the ABA
concentration required for half-maximal inhibition of
phosphatase activity (IC50) and the effect of PYL-PP2C
ratio on the ABA IC50 (Szostkiewicz et al., 2010). In
general, higher PYL-PP2C ratios favored inhibition
at low levels of ABA, while lower PYL-PP2C ratios
allowed greater phosphatase activity. Antoni et al.
(2012) observed that HAI1 activity could be inhibited
to some extent by both dimeric and monomeric PYLs,
at least when no other proteins were present to compete for interaction with the PP2C. However, the IC50
values for the dimeric PYLs were very high, indicating
the HAI1 was resistant to inhibition by these PYLs.
Although their study did not examine the full range of
PYLs, the results were consistent with ours in that
PYL5 and PYL8 were most effective in inhibiting HAI1
activity and gave the strongest interactions in our
yeast two-hybrid experiments. The phosphatase activity assays of Antoni et al. (2012) were constructed
with a 4:1 or 10:1 PYL:PP2C ratio, and it was pointed
out that a 4:1 PYL:PP2C ratio gave a more realistic
indication of PP2C regulation than a 100:1 PYL:PP2C
ratio, which caused severalfold greater inhibition
of phosphatase activity (Hao et al., 2011). Our data
showing several hundred fold induction of HAI1 expression at the same time that expression of its interacting PYLs was mostly down-regulated suggest that
even a 4:1 PYL:HAI1 ratio may be unlikely to exist in
vivo. It has also been demonstrated that HAB1 mutations that weakened its interaction with PYR1 allowed
it to remain active and dephosphorylate the SnRK2
OST1 even in the presence of PYR1 and ABA concentrations that inhibited the activity of wild-type HAB1
(Dupeux et al., 2011). Wild-type HAI1 already has such
weakened interaction. Thus, the combination of weak
PYL interaction and high expression indicates that PYL
regulation may not be a major determinant of HAI1
function at low Cw. It was also suggested that the ABAindependent PP2C interactions of the monomeric PYLs
were less effective in PP2C regulation than PP2C-ABAPYL ternary complexes (Antoni et al., 2012). The strong
interaction of the monomeric PYLs, particularly PYL7,
with AIP1 and HAI3 observed in our experiments
suggests that this may not always be the case. Phosphatase activity assays using the monomeric PYLs
with AIP1 or HAI3 will be of interest for future studies.
This model of limited PYL regulation, which allows
the HAI PP2Cs, particularly HAI1, to remain active
during low Cw, also suggests the importance of their
dephosphorylation substrate proteins as regulators of
the low-Cw response. Structural studies have shown
that PYLs and SnRK2 kinases mimic each other in their
binding to PP2Cs (Soon et al., 2012). Thus, sequence
differences in the HAI PP2Cs compared with other
clade A PP2Cs (Supplemental Fig. S11) in and around
the amino acids critical for PYL interaction may also

indicate different specicity in their interactions with
SnRK2s or other unidentied substrates. Other differences between the HAI PP2Cs compared with other
clade A PP2Cs are found in the ABA box, which tethers
some SnRK2s and PP2Cs (such as HAB1) to each other
(Soon et al., 2012), and in the motif that determines
SnRK3 interaction specicity of ABI1 and ABI2 (Ohta
et al., 2003; Supplemental Fig. S11). These factors indicate that identifying dephosphorylation targets of
the HAI PP2Cs will be key to understanding their
unique function in low-Cw signaling.
Unique Stress Physiology Function of the HAI PP2Cs
Osmoregulatory solute accumulation (osmotic adjustment) has been well studied by crop physiologists,
who have found substantial genetic variability in this
trait (Morgan, 1984, 1991). The increased solute accumulation of hai1, aip1, and hai3 establish the HAI PP2Cs
as one of the few molecular components known to
regulate osmotic adjustment. Interestingly, one of the
few other genes shown to regulate osmotic adjustment
are group C mitogen-activated protein kinases of cotton (Gossypium hirsutum), whose overexpression could
increase both Pro and osmoregulatory solute accumulation (Zhang et al., 2011). It is tempting to speculate that these phosphatases and kinases may directly
antagonize each other at the molecular level.
Our data also raise the question of why the negative
regulation mediated by the HAI PP2Cs is maintained
when it may seem that maximizing osmotic adjustment
and Pro would be a better drought-adaptive strategy
that would emerge through natural selection. An explanation is suggested by the observation that hai1-2
had opposing effects on a number of abiotic stressassociated genes (up-regulated in hai1-2 relative to the
wild type) and defense-related genes (down-regulated
in hai1-2). Antagonism between ABA/abiotic stress
signaling versus biotic stress/defense signaling is an
emerging topic in plant-environment interaction (Yasuda
et al., 2008; Huang et al., 2010; Kim et al., 2011). Fitness
tradeoffs between pathogen defense and drought response would suggest that maximizing drought responses may not always be best for overall adaptation
and that negative regulation, such as that mediated by
HAI1 and other PP2Cs, is important to balance the two
responses.
MATERIALS AND METHODS

Plant Material and Stress Treatments
T-DNA insertion lines of Arabidopsis (Arabidopsis thaliana) were obtained
from the Arabidopsis Biological Resource Center, and primers used for genotyping are given in Supplemental Table S9. Double and triple mutants were
generated by crossing hai1-2 to aip1-1 and then crossing hai1-2aip1-1 to hai3-1.
hai3-1 was further analyzed by twice backcrossing to the wild type and
selecting homozygous plants for Pro analysis. hai1-2 was also used for
transgenic complementation. The open reading frame of HAI1 was amplied

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Bhaskara et al.
(primers are given in Supplemental Table S9), cloned into pDONR207,

transferred to pGWB442 (Nakagawa et al., 2007) to generate 35S:YFP-HAI1,
and transformed into hai1-2.
For seedling growth and stress treatments, sterilized seeds were plated on
one-half-strength Murashige and Skoog medium with MES buffer (pH 5.7) but
without added sugar. Plates were stratied for 4 d at 4C, and the seedlings
were grown by placing the plates vertically in a growth chamber (25C,
continuous light at 80100 mmol photons m22 s21). Seven-day-old seedlings
were transferred to PEG-infused plates (Verslues et al., 2006) to impose lowCw stress. Alternatively, seedlings were transferred to plates containing NaCl
or S(+)-ABA added to the medium after sterilization. For germination or
cotyledon emergence assays, approximately, 100 seeds per genotype were
sown on plates with or without S(+)-ABA, radicle emergence was scored after
4 d, and green cotyledon emergence was examined after 5 or 8 d.
For leaf water loss experiments, plants were grown under long-day conditions, and fully expanded rosette leaves were collected from 4-week-old
plants and weighed over the course of 8 h to monitor water loss. For soil
drying experiments, plants were grown under short-day conditions in a growth
chamber, and the normal potting mixture was supplemented with 40% ne
sand to facilitate even soil drying. Watering was stopped after 30 d of growth,
and measurements were conducted over the subsequent 6 to 12 d of water
withholding.
Physiological Assays
For Pro measurement, seedlings were collected 96 h after transfer to various
Cw treatments and analyzed by ninhydrin assay adapted to a 96-well plate
format (Bates et al., 1973; Verslues, 2010). For Cs measurement, seedling or leaf
samples were frozen, macerated with a microfuge tube pestle, and centrifuged
to pellet insoluble material. Cs of the cell sap was measured using a Wescor
Psypro system with c-52 sample chambers. Cw of agar or soil medium collected at the same time was also measured. Seedling fresh weight was measured by weighing groups of six to 10 seedlings and calculating the per
seedling weight. RWC was measured by detaching fully expanded leaves,
weighing, oating on water for 9 to 10 h, reweighing, and drying overnight in
a 60C oven. RWC was calculated as (fresh weight 2 dry weight)/hydrated
weight 2 dry weight) 3 100.
ABA analysis was performed by extracting freeze-dried seedlings (50200
mg fresh weight) in 80% methanol with 25 pmol of [D6]ABA (Plant Biotechnology Institute) as an internal standard. Extracts were passed through a C18
solid-phase extraction cartridge (Supelco), evaporated to dryness, resuspended in diethyl ether:methanol (9:1), and derivatized by the addition of
trimethylsilydiazomethane (Sigma). After derivatization, remaining trimethyldiazomethane was destroyed by the addition of 0.5 M acetic acid in hexane
(Schmelz et al., 2003). The samples were then evaporated, resuspended in a
small volume of ethyl acetate, injected onto a VF-14MS (Varian/Agilent) column,
and analyzed by tandem mass spectrometry. Methanol chemical ionization
was used to generate precursor ions (261 mass-to-charge ratio [m/z] for ABA
and 267 m/z for [D6]ABA). Daughter ions of 229 m/z (ABA) and 233 + 234 m/z
([D6]ABA) were used for quantication (Mller et al., 2002). The ABA content of
samples was quantied by a standard curve over 2 to 60 pmol of ABA prepared
using the ratio of the 229 and 233 + 234 peak areas.
based on Actin8 expression. All primers used are given in Supplemental

Table S9. The expression of Pro metabolism genes, NCED3 and COR15A,
was quantied using TaqMan probes and standard curves for each gene as
described previously (Sharma and Verslues, 2010).
Yeast Two-Hybrid Analysis

Constructs for yeast two-hybrid analysis were prepared by PYL and PP2C
amplication from either Col wild-type DNA (for PYLs lacking introns) or
from full-length complementary DNA clones obtained from the Arabidopsis
Biological Resource Center (Supplemental Table S9), cloning into pDONOR207, and transfer to yeast two-hybrid destination vectors by Gateway
reaction. Yeast two-hybrid screening was performed using the ProQuest twohybrid system (Invitrogen). Yeast strain MaV203a was transformed with
pEXP32-HAI1 or N HAI1 (lacking amino acids 1104), N AIP1 (lacking
amino acids 1118), or HAI3 or N HAB1 (lacking amino acids 1178;
Santiago et al., 2009b) bait plasmid and pEXP22-PYL prey plasmid using the
lithium acetate method. Primary transformants were selected for growth on
Trp/Leu dropout plates. Trp-Leu+ colonies were analyzed for b-galactosidase
activity by colony-lift lter assay. Quantitation of interaction strength was
performed using chlorophenol red-b-D-galactopyranoside (CPRG) assay following the manufacturers instruction (Invitrogen). At least three different
colonies per two-hybrid pair were grown in selective minimal medium
overnight at 30C and then were inoculated in 5 mL of yeast extract-peptoneadenine-dextrose (YPAD) medium with or without ABA until the A600 reached
1.0 to 1.5. Cells were collected and washed with cold water. The cell pellet was
resuspended in 100 mL of buffer 1 (100 mM HEPES,154 mM NaCl, 4.5 mM L-Asp,
0.1 g L21 bovine serum albumin, and 500 mL L21 Tween 20, pH 7.257.30).
Cells were broken using a bead beater and acid-washed 0.5-mm glass beads.
The CPRG reaction was started by mixing lysed cells with 900 mL of buffer 2
(27.1 mg of CPRG in 20 mL of buffer 1), and A574 was measured.
For western-blot detection of fusion proteins in yeast, an overnight culture
in selective medium was subcultured in YPAD for 4 to 6 h, and equal amounts
of cells were collected based on A600. Proteins were extracted (Kushnirov,
2000), separated by SDS-PAGE, electroblotted onto polyvinylidene diuoride
membranes, probed with antibody against the GAL4 DNA-binding domain
(Abcam) and horseradish peroxidase-conjugated secondary antibody (Abcam),
and developed using TMA-6 reagent (Lumigen).
Statistical Analysis
Data were analyzed by ANOVA (using Sigma Plot 11) or t test as indicated
in the text or gure legends.
Complete data from the microarray analysis of the Col wild type and hai1-2
under control conditions and after 96 h of low-Cw treatment are available in
the National Center for Biotechnology Information Gene Expression Omnibus
under accession number GSE35258.
Supplemental Data
The following materials are available in the online version of this article.
Gene Expression
Supplemental Figure S1. Clade A PP2C T-DNA lines.
Total RNA was extracted from control or low-Cw-treated seedlings using

RNeasy plant mini kits (Qiagen). Microarray analysis was conducted at the
Affymetrix Gene Expression Service Laboratory of Academia Sinica using
Arabidopsis ATH1 chips (Affymetrix) and standard protocols. Three biological replicates were used for all microarray analysis. Data were analyzed using
GeneSpring software. A Benjamini-Hochberg corrected value of P # 0.05 and
change greater than 1.5-fold were used to dene genes differentially expressed
between the wild type and hai1-2. Coexpression clustering was done using the
Maccu toolbox (Lin et al., 2011) based on data of 3,800 slides downloaded
from the NASCarrays database (Craigon et al., 2004). GO enrichment was
computed using the TopGO elim method (Alexa et al., 2006) using GOBU with
its MultiView plugin (Lin et al., 2006).
For quantitative RT-PCR analysis, RNA (typically 1 mg) from at least three
independently collected samples was reverse transcribed using SuperScript III
(Invitrogen). Quantitative RT-PCR was performed using the KAPA SYBR
FAST qPCR kit (Kapa Biosystems), and fold change in expression was calculated by the comparative cycle threshold method following normalization
Supplemental Figure S2. Low water potential-induced Pro accumulation

of HAI PP2C double and triple mutants.
392
Supplemental Figure S3. Salt stress-induced Pro accumulation of HAI

PP2C double and triple mutants.
Supplemental Figure S4. Osmotic potential and seedling fresh weight of
HAI PP2C double and triple mutants after low water potential treatment.
Supplemental Figure S5. Wild-type (Col) and hai1-2 plants during soil
drying.
Supplemental Figure S6. Coexpression clusters formed from genes downregulated in hai1-2 relative to the Col wild type.
Supplemental Figure S7. Quantitative RT-PCR analysis of Pro metabolism
genes shows similar expression in the wild type and hai1-2.
Supplemental Figure S8. Conrmation of ABA-hypersensitive germination in mutants of the ABA-signaling Clade A PP2Cs.

Supplemental Figure S9. ABA-induced Pro accumulation does not differ in

HAI PP2C single mutants and is only slightly increased in the triple mutant.
Supplemental Figure S10. Low water potential-induced ABA accumulation of Clade A PP2C mutants.
Supplemental Figure S11. Alignment of the C-terminal regions containing
the active site and PYL-interaction sites of the HAI PP2Cs, ABI1, ABI2,
and HAB1 shows sequence differences which may affect PYL interaction
and substrate specicity of the HAI PP2Cs.
Supplemental Figure S12. Additional yeast two-hybrid assays and western-blot assays with HAB1 and HAI1.
Supplemental Table S1. Genes up-regulated in Col wild-type seedlings at
low water potential.
Supplemental Table S2. Genes down-regulated in Col wild-type seedlings
at low water potential.
Supplemental Table S3. Genes up-regulated in hai1-2 relative to the wild
type in control (high water potential).
Supplemental Table S4. Genes down-regulated in hai1-2 relative to the
wild type in control (high water potential).
Supplemental Table S5. Genes up-regulated in hai1-2 relative to the wild
type at low water potential.
Supplemental Table S6. Genes down-regulated in hai1-2 relative to the
wild type at low water potential.
Supplemental Table S7. GO enrichment analysis of genes up-regulated in
hai1-2 relative to the wild type under either unstressed control or low
water potential treatments.
Supplemental Table S8. GO enrichment analysis of genes down-regulated
in hai1-2 relative to the wild type under either unstressed control or low
water potential treatments.
Supplemental Table S9. Primer sequences used in this study.
ACKNOWLEDGMENTS
Affymetrix GeneChip assays were performed by the Affymetrix Gene
Expression Service Laboratory (http://ipmb.sinica.edu.tw/affy/) supported
by Academia Sinica. We thank Tsu-Hao Yang for assistance with yeast twohybrid and western-blot experiments, Wendar Lin and the bioinformatics core
facility of the Institute of Plant and Microbial Biology for the coexpression
clustering and GO enrichment analyses, Min-Yan Kuo for assistance with
microarray analysis, Mei-Jane Fang for assistance with microscopy, Na Lin
for general laboratory assistance, and Wendy Hwang-Verslues for critical
reading of the manuscript.
Received June 22, 2012; accepted July 20, 2012; published July 24, 2012.
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Unique Drought Resistance Functions of the

Highly ABA-Induced Clade A Protein Phosphatase 2Cs1[W][OA]

Plants have well-developed sensing and signal

Hypersensitive Germination1 (AHG1), AHG3/AtPP2CA,

Downloaded from www.plantphysiol.org on June 16, 2014 - Published by www.plant.org

and Xenopus laevis oocytes (Fujii et al., 2009; Brandt

compensated by an accumulation of solutes inside the

We collected transferred DNA (T-DNA) mutants of

Downloaded from www.plantphysiol.org on June 16, 2014 - Published by www.plant.org

Function of HAI Clade A PP2Cs in Drought Stress

Figure 1. HAI PP2Cs are negative regulators of

negative mutants had reduced Pro accumulation

Plant Physiol. Vol. 160, 2012

Downloaded from www.plantphysiol.org on June 16, 2014 - Published by www.plant.org

None of the HAI PP2C mutants had increased Pro in

HAI1, AIP1, and HAI3 Are Negative Regulators of

Plant Physiol. Vol. 160, 2012

Downloaded from www.plantphysiol.org on June 16, 2014 - Published by www.plant.org

Function of HAI Clade A PP2Cs in Drought Stress

hab2-1 did not differ from wild-type Cs at any severity

HAI1 Is a Negative Regulator of Osmotic Adjustment

We planted mutant and wild-type plants together

Figure 3. hai1-2 has increased osmotic adjustment during soil drying,

Plant Physiol. Vol. 160, 2012

Downloaded from www.plantphysiol.org on June 16, 2014 - Published by www.plant.org

(Fig. 3A). Leaf relative water content (RWC) declined

HAI PP2C Mutants Have Little Effect on Leaf Water Loss

hai1-2 Has Enhanced Expression of Dehydration-Protective

We conducted microarray analysis of hai1-2, as it

and ABA signaling were also signicantly enriched.

HAI PP2C Double and Triple Mutants Have Differential

To further dene unique and overlapping functions

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Function of HAI Clade A PP2Cs in Drought Stress

double and triple mutants of other clade A PP2Cs

ABA concentrations (Fig. 6C). ABA hypersensitivity

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Figure 5. HAI PP2C double and triple mutants

HAI PP2Cs did uncover ABA hypersensitivity but also

HAI PP2Cs Have a Distinct Pattern of PYL Interaction

One basis for the unique physiological function of

HAI1 with those of HAB1, whose PYL interactions

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Function of HAI Clade A PP2Cs in Drought Stress

was the lack of HAI1 interaction with PYL7, which

7B). These experiments used an N-terminal AIP1

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AIP1, or HAI3 unless ABA was added. PYL7 had weak

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Function of HAI Clade A PP2Cs in Drought Stress

HAB1 (Supplemental Fig. S12A). We also constructed

Our microarray analysis indicated that the three HAI

expression of PYL7 and PYL10 indicates that they

Our analysis found both some similarity as well

Plant Physiol. Vol. 160, 2012

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genes, including dehydrins and LEAs. Overall, the data

osmoregulatory solute accumulation. The specic PYL

Recent studies have continued to add complexity

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